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1.
Methods Enzymol ; 699: 231-263, 2024.
Article in English | MEDLINE | ID: mdl-38942505

ABSTRACT

Terpenes are a diverse class of natural products which have long been sought after for their chemical properties as medicine, perfumes, and for food flavoring. Computational docking studies of terpene mechanisms have been a challenge due to the lack of strong directing groups which many docking programs rely on. In this chapter, we dive into our computational method Terdockin (Terpene-Docking) as a successful methodology in modeling terpene synthase mechanisms. This method could also be used as inspiration for any multi-ligand docking project.


Subject(s)
Alkyl and Aryl Transferases , Catalytic Domain , Molecular Docking Simulation , Terpenes , Molecular Docking Simulation/methods , Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/metabolism , Terpenes/chemistry , Terpenes/metabolism , Ligands
2.
Int J Mol Sci ; 23(6)2022 Mar 14.
Article in English | MEDLINE | ID: mdl-35328548

ABSTRACT

Trace amine-associated receptor 5 (TAAR5) is a G protein-coupled receptor that belongs to the TAARs family (TAAR1-TAAR9). TAAR5 is expressed in the olfactory epithelium and is responsible for sensing 3-methylamine (TMA). However, recent studies showed that TAAR5 is also expressed in the limbic brain regions and is involved in the regulation of emotional behaviour and adult neurogenesis, suggesting that TAAR5 antagonism may represent a novel therapeutic strategy for anxiety and depression. We used the AtomNet® model, the first deep learning neural network for structure-based drug discovery, to identify putative TAAR5 ligands and tested them in an in vitro BRET assay. We found two mTAAR5 antagonists with low to submicromolar activity that are able to inhibit the cAMP production induced by TMA. Moreover, these two compounds also inhibited the mTAAR5 downstream signalling, such as the phosphorylation of CREB and ERK. These two hits exhibit drug-like properties and could be used to further develop more potent TAAR5 ligands with putative anxiolytic and antidepressant activity.


Subject(s)
Receptors, G-Protein-Coupled , Signal Transduction , Ligands , Neural Networks, Computer , Olfactory Mucosa
3.
Biochemistry ; 59(47): 4507-4515, 2020 12 01.
Article in English | MEDLINE | ID: mdl-33182997

ABSTRACT

Terpenes make up the largest class of natural products, with extensive chemical and structural diversity. Diterpenes, mostly isolated from plants and rarely prokaryotes, exhibit a variety of important biological activities and valuable applications, including providing antitumor and antibiotic pharmaceuticals. These natural products are constructed by terpene synthases, a class of enzymes that catalyze one of the most complex chemical reactions in biology: converting simple acyclic oligo-isoprenyl diphosphate substrates to complex polycyclic products via carbocation intermediates. Here we obtained the second ever crystal structure of a class II diterpene synthase from bacteria, tuberculosinol pyrophosphate synthase (i.e., Halimadienyl diphosphate synthase, MtHPS, or Rv3377c) from Mycobacterium tuberculosis (Mtb). This enzyme transforms (E,E,E)-geranylgeranyl diphosphate into tuberculosinol pyrophosphate (Halimadienyl diphosphate). Rv3377c is part of the Mtb diterpene pathway along with Rv3378c, which converts tuberculosinol pyrophosphate to 1-tuberculosinyl adenosine (1-TbAd). This pathway was shown to exist only in virulent Mycobacterium species, but not in closely related avirulent species, and was proposed to be involved in phagolysosome maturation arrest. To gain further insight into the reaction pathway and the mechanistically relevant enzyme substrate binding orientation, electronic structure calculation and docking studies of reaction intermediates were carried out. Results reveal a plausible binding mode of the substrate that can provide the information to guide future drug design and anti-infective therapies of this biosynthetic pathway.


Subject(s)
Alkyl and Aryl Transferases/chemistry , Diterpenes/metabolism , Models, Molecular , Mycobacterium tuberculosis/enzymology , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cloning, Molecular , Crystallography, X-Ray , Cyclization/genetics , Diterpenes/chemistry , Molecular Docking Simulation , Mycobacterium tuberculosis/genetics
4.
ACS Catal ; 8(4): 3322-3330, 2018 Apr 06.
Article in English | MEDLINE | ID: mdl-30034923

ABSTRACT

Terpene synthases comprise a family of enzymes that convert acyclic oligo-isoprenyl diphosphates to terpene natural products with complex, polycyclic carbon backbones via the generation and protection of carbocation intermediates. To accommodate this chemistry, terpene synthase active sites generally are lined with alkyl and aromatic, i.e., nonpolar, sidechains. Predicting the correct, mechanistically relevant binding modes for entire terpene synthase reaction pathways remains an unsolved challenge. Here we describe a method for identifying such modes: TerDockin, a series of protocols to predict the orientation of carbon skeletons of substrates and derived carbocations relative to the bound diphosphate group in terpene synthase active sites. Using this recipe for bornyl diphosphate synthase, we have predicted binding modes that are consistent with all current experimental observations, including the results of isotope labeling experiments and known stereoselectivity. In addition, the predicted binding modes recapitulate key findings of a seminal study involving more computationally demanding QM/MM molecular dynamics methods on part of this pathway. This work illustrates the value of the TerDockin approach as a starting point for more involved calculations and sets the stage for the rational engineering of this family of enzymes.

5.
ACS Catal ; 8(4): 3133-3137, 2018 Apr 06.
Article in English | MEDLINE | ID: mdl-29713562

ABSTRACT

Sclareol synthase from Salvia sclarea (SsSS) naturally acts on 8α-hydroxy-copalyl diphosphate (1), stereoselectively adding water to produce (13R)-sclareol (2a), and similarly yields hydroxylated products with manifold other such bicyclic diterpene precursors. Here a key residue for this addition of water was identified. Strikingly, substitution with glutamine switches stereochemical outcome with 1, leading to selective production of (13S)-sclareol (2b). Moreover, changes to the stereospecificity of water addition with the structurally closely-related substrate copalyl diphosphate (4) could be accomplished with alternative substitutions. Thus, this approach is expected to provide biosynthetic access to both epimers of 13-hydroxylated derivatives of manifold labdane-related diterpenes.

6.
ACS Chem Biol ; 12(9): 2465-2473, 2017 09 15.
Article in English | MEDLINE | ID: mdl-28820936

ABSTRACT

The rapidly growing appreciation of enzymes' catalytic and substrate promiscuity may lead to their expanded use in the fields of chemical synthesis and industrial biotechnology. Here, we explore the substrate promiscuity of enoyl-acyl carrier protein reductases (commonly known as FabI) and how that promiscuity is a function of inherent reactivity and the geometric demands of the enzyme's active site. We demonstrate that these enzymes catalyze the reduction of a wide range of substrates, particularly α,ß-unsaturated aldehydes. In addition, we demonstrate that a combination of quantum mechanical hydride affinity calculations and molecular docking can be used to rapidly categorize compounds that FabI can use as substrates. The results here provide new insight into the determinants of catalysis for FabI and set the stage for the development of a new assay for drug discovery, organic synthesis, and novel biocatalysts.


Subject(s)
Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/metabolism , Plasmodium falciparum/enzymology , Protozoan Proteins/metabolism , Catalytic Domain , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/chemistry , Humans , Malaria, Falciparum/parasitology , Molecular Docking Simulation , Plasmodium falciparum/chemistry , Plasmodium falciparum/metabolism , Protozoan Proteins/chemistry , Substrate Specificity
7.
ACS Omega ; 2(7): 3308-3314, 2017 Jul 31.
Article in English | MEDLINE | ID: mdl-31457655

ABSTRACT

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs shows that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. These results are directly applicable to rational enzyme design and engineering.

8.
ACS Med Chem Lett ; 6(3): 308-12, 2015 Mar 12.
Article in English | MEDLINE | ID: mdl-25815151

ABSTRACT

The bacterial cell division protein FtsZ is one of many potential targets for the development of novel antibiotics. Recently, zantrin Z3 was shown to be a cross-species inhibitor of FtsZ; however, its specific interactions with the protein are still unknown. Herein we report the synthesis of analogues that contain a more tractable core structure and an analogue with single-digit micromolar inhibition of FtsZ's GTPase activity, which represents the most potent inhibitor of Escherichia coli FtsZ reported to date. In addition, the zantrin Z3 core has been converted to two potential photo-cross-linking reagents for proteomic studies that could shed light on the molecular interactions between FtsZ and molecules related to zantrin Z3.

9.
ACS Med Chem Lett ; 3(10): 818-822, 2012 Aug 28.
Article in English | MEDLINE | ID: mdl-23119123

ABSTRACT

The synthesis and antimicrobial activity heterocyclic analogs of the diterpenoid totarol are described. An advanced synthetic intermediate with a ketone on the A-ring is used to attach fused heterocycles and a carbon-to-nitrogen atom replacement is made on the B-ring by de novo synthesis. A-ring analogs with an indole attached exhibit, for the first time, enhanced antimicrobial activity relative to the parent natural product. Preliminary experiments demonstrate that the indole analogs do not target the bacterial cell division protein FtsZ as had been hypothesized for totarol.

10.
ACS Chem Biol ; 7(11): 1918-28, 2012 Nov 16.
Article in English | MEDLINE | ID: mdl-22958099

ABSTRACT

FtsZ is a guanosine triphosphatase (GTPase) that mediates cytokinesis in bacteria. FtsZ is homologous in structure to eukaryotic tubulin and polymerizes in a similar head-to-tail fashion. The study of tubulin's function in eukaryotic cells has benefited greatly from specific and potent small molecule inhibitors, including colchicine and taxol. Although many small molecule inhibitors of FtsZ have been reported, none has emerged as a generally useful probe for modulating bacterial cell division. With the goal of establishing a useful and reliable small molecule inhibitor of FtsZ, a broad biochemical cross-comparison of reported FtsZ inhibitors was undertaken. Several of these molecules, including phenolic natural products, are unselective inhibitors that seem to derive their activity from the formation of microscopic colloids or aggregates. Other compounds, including the natural product viriditoxin and the drug development candidate PC190723, exhibit no inhibition of GTPase activity using protocols in this work or under published conditions. Of the compounds studied, only zantrin Z3 exhibits good levels of inhibition, maintains activity under conditions that disrupt small molecule aggregates, and provides a platform for exploration of structure-activity relationships (SAR). Preliminary SAR studies have identified slight modifications to the two side chains of this structure that modulate the inhibitory activity of zantrin Z3. Collectively, these studies will help focus future investigations toward the establishment of probes for FtsZ that fill the roles of colchicine and taxol in studies of tubulin.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteria/enzymology , Bacterial Proteins/antagonists & inhibitors , Cytoskeletal Proteins/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Bacillus subtilis/drug effects , Bacillus subtilis/enzymology , Bacteria/drug effects , Bacterial Infections/drug therapy , Bacterial Proteins/metabolism , Cytoskeletal Proteins/metabolism , Escherichia coli/drug effects , Escherichia coli/enzymology , Humans , Pyridines/chemistry , Pyridines/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymology , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacology
11.
Bioorg Med Chem ; 18(16): 5995-6005, 2010 Aug 15.
Article in English | MEDLINE | ID: mdl-20643554

ABSTRACT

The insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase (RTK) involved in all stages of the development and propagation of breast and other cancers. The inhibition of IGF-1R by small molecules remains a promising strategy to treat cancer. Herein, we explore SAR around previously characterized lead compound (1), which is an aryl-heteroaryl urea (AHU) consisting of 4-aminoquinaldine and a substituted aromatic ring system. A library of novel AHU compounds was prepared based on derivatives of the 4-aminoquinoline heterocycle (including various 2-substituted derivatives, and naphthyridines). The compounds were screened for in vitro inhibitory activity against IGF-1R, and several compounds with improved activity (3-5 microM) were identified. Furthermore, a computational docking study was performed, which identifies a fairly consistent lowest energy mode of binding for the more-active set of inhibitors in this series, while the less-active inhibitors do not adopt a consistent mode of binding.


Subject(s)
Aminoquinolines/chemistry , Aminoquinolines/pharmacology , Receptors, Somatomedin/antagonists & inhibitors , Receptors, Somatomedin/metabolism , Urea/chemistry , Urea/pharmacology , Aminoquinolines/chemical synthesis , Humans , Models, Molecular , Protein Binding , Receptors, Somatomedin/chemistry , Urea/chemical synthesis
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